Stability of high cell density brewery fermentations during serial repitching

The volumetric productivity of the beer fermentation process can be increased by using a higher pitching rate (i.e. higher inoculum size). However, the decreased yeast net growth observed in these high cell density brewery fermentations can adversely affect the physiological stability throughout subsequent yeast generations. Therefore, different O₂ conditions (wort aeration and yeast preoxygenation) were applied to high cell density fermentation and eight generations of fermentations were evaluated together with conventional fermentations. Freshly propagated high cell density populations adapted faster to the fermentative conditions than normal cell density populations. Preoxygenating the yeast was essential for the yeast physiological and beer flavor compound stability of high cell density fermentations during serial repitching. In contrast, the use of non-preoxygenated yeast resulted in inadequate growth which caused (1) insufficient yield of biomass to repitch all eight generations, (2) a 10% decrease in viability, (3) a moderate increase of yeast age, (4) and a dramatic increase of the unwanted flavor compounds acetaldehyde and total diacetyl during the sequence of fermentations. Therefore, to achieve sustainable high cell density fermentations throughout the economical valuable process of serial repitching, adequate yeast growth is essential.     

Impact of pitching rate on yeast fermentation performance and beer flavour

The volumetric productivity of the beer fermentation process can be increased by using a higher pitching rate (i.e. higher inoculum size). However, the impact of the pitching rate on crucial fermentation and beer quality parameters has never been assessed systematically. In this study, five pitching rates were applied to lab-scale fermentations to investigate its impact on the yeast physiology and beer quality. The fermentation rate increased significantly and the net yeast growth was lowered with increasing pitching rate, without affecting significantly the viability and the vitality of the yeast population. The build-up of unsaturated fatty acids in the initial phase of the fermentation was repressed when higher yeast concentrations were pitched. The expression levels of the genes HSP104 and HSP12 and the concentration of trehalose were higher with increased pitching rates, suggesting a moderate exposure to stress in case of higher cell concentrations. The influence of pitching rate on aroma compound production was rather limited, with the exception of total diacetyl levels, which strongly increased with the pitching rate. These results demonstrate that most aspects of the yeast physiology and flavour balance are not significantly or negatively affected when the pitching rate is changed. However, further research is needed to fully optimise the conditions for brewing beer with high cell density populations.     

Isolation and utilization of indigenous yeast strain for brewing of millet beer (OYOKPO)

Three yeast strains were isolated from a spontaneously fermented native millet (Pennisetum typhoideum) malt beer (Oyokpo). One of the yeast isolates found to have the most highly fermenting capacity was characterised and identified as a strain of Saccharomyces cerevisiae. The yeast was then utilised as the pitching yeast in a subsequent controlled fermentation of millet wort at 20°C for 120 hours. Bitter leaf (Vernonia amagdalina) extract was used as the bittering and flavouring agent. The Oyokpo beer sample produced under these conditions was found to possess both chemical and organoleptic qualities comparable to some extent, to the conventional barley malt beer. At the end of fermentation, the pH, specific gravity, alcohol content, reducing sugar content and protein content of the beer were 4.11, 1.0308, 2.81% (v/v), 4.00 (mg/ml) and 0.84 (mg/ml) respectively.     

Yeast responses to stresses associated with industrial brewery handling

During brewery handling, production strains of yeast must respond to fluctuations in dissolved oxygen concentration, pH, osmolarity, ethanol concentration, nutrient supply and temperature. Fermentation performance of brewing yeast strains is dependent on their ability to adapt to these changes, particularly during batch brewery fermentation which involves the recycling (repitching) of a single yeast culture (slurry) over a number of fermentations (generations). Modern practices, such as the use of high-gravity worts and preparation of dried yeast for use as an inoculum, have increased the magnitude of the stresses to which the cell is subjected. The ability of yeast to respond effectively to these conditions is essential not only for beer production but also for maintaining the fermentation fitness of yeast for use in subsequent fermentations. During brewery handling, cells inhabit a complex environment and our understanding of stress responses under such conditions is limited. The advent of techniques capable of determining genomic and proteomic changes within the cell is likely vastly to improve our knowledge of yeast stress responses during industrial brewery handling.     

A comparative study on physiological activities of lager and ale brewing yeasts under different gravity conditions

High gravity (HG) or very high gravity (VHG) brewing has become popular in modern breweries due to its economic and product quality advantages. However, there are the negative impacts such as the fermentation performance of brewer??s yeast in HG or VHG wort, which are closely related to changes in cell physiological activity. In the present study, 3 kinds of worts, with different gravities, were used to examine the systematic effects on fermentation performance and physiological activity of lager yeast FBY009505 (Saccharomyces pastorianus) and ale yeast FBY0099 (Saccharomyces cerevisiae), as well as the resulting beer flavor. Results showed that the responses of FBY009505 and FBY0099 to the HG or VHG worts were similar. The specific fermentation rate and viability of cropped yeast of FBY009505 and FBY0099 were decreased with increasing wort gravity. The increased wort gravity resulted in the increase of energy charge and the decrease of ??-glucosides transport rate and glycolytic enzyme activities. Moreover, the environmental stresses in the HG or VHG wort showed a higher inhibitory activity against ??-glucoside transport than glycolytic enzymes. The content of intracellular trehalose and glycerol of FBY009505 and FBY0099 increased with the increase in wort gravity. The results from this study provided a potential means to systematically understand the physiology of brewer??s yeast under HG or VHG conditions.     

Distribution of Bacterial Contaminants in a South African Lager Brewery

Bacteria isolated from contaminated pitching yeast, fermenting wort and beer samples from a South African lager brewery over a one-year period were tentatively identified by an improved, rapid diagnostic procedure as pediococci (41%), homofermentative lactobacilli (5%), heterofermentative lactobacilli (9%), Acetobacter spp. (7%), Gluconobacter spp. (13%) and Hafnia protea (25%). Pediococci and lactobacilli dominated samples taken from fermentation, storage and 'bright' beer tanks but were absent from pitched wort samples, from collection vessels and the single pitching yeast sample investigated. Acetic acid bacteria and H. protea were widely distributed in collection vessel, fermentation and storage tank samples, and H. protea was isolated from recycled pitching yeast.     

Proteases supplementation to high gravity worts enhances fermentation performance of brewer's yeast

Nitrogen limitation, particularly prevailing in the case of high gravity beer brewing, results in poor yeast viability and even stuck or sluggish fermentations. Although wort contains abundant proteins and longer chain peptides, brewer's yeast does not assimilate them due to the fact that cells hardly secrete proteases during fermentation. The objective of this study was to investigate the possibility for utilizing unavailable nitrogen from two types of high gravity worts (20 °P and 24 °P) by adding three food-grade commercial proteases (Neutrase, Flavorzyme and Protamex) at the beginning of fermentations, respectively. Results showed that proteases supplementation significantly increased the FAN level and thus the amount of cell suspension in the later stages of fermentations (ca. 10 days later for 20 °P and 25 days later for 24 °P) (p < 0.05). Among the studied three proteases, we found that fermentations with Flavorzyme supplementation exhibited the best fermentation performance in terms of significantly improved wort fermentability, higher ethanol yield and flavor volatiles formation (p < 0.05). Furthermore, the foam of final beers produced by adding proteases was as stable as that of the control at each of the corresponding gravities.     

The role of oxygen in yeast metabolism during high cell density brewery fermentations

The volumetric productivity of the beer fermentation process can be increased by using a higher pitching rate (i.e., higher inoculum size). However, the decreased yeast net growth observed in these high cell density fermentations can have a negative impact on the physiological stability throughout subsequent yeast generations. The use of different oxygen conditions (wort aeration, wort oxygenation, yeast preoxygenation) was investigated to improve the growth yield during high cell density fermentations and yeast metabolic and physiological parameters were assessed systematically. Together with a higher extent of growth (dependent on the applied oxygen conditions), the fermentation power and the formation of unsaturated fatty acids were also affected. Wort oxygenation had a significant decreasing effect on the formation of esters, which was caused by a decreased expression of the alcohol acetyl transferase gene ATF1, compared with the other conditions. Lower glycogen and trehalose levels at the end of fermentation were observed in case of the high cell density fermentations with oxygenated wort and the reference fermentation. The expression levels of BAP2 (encoding the branched chain amino acid permease), ERG1 (encoding squalene epoxidase), and the stress responsive gene HSP12 were predominantly influenced by the high cell concentrations, while OLE1 (encoding the fatty acid desaturase) and the oxidative stress responsive genes SOD1 and CTT1 were mainly affected by the oxygen availability per cell. These results demonstrate that optimisation of high cell density fermentations could be achieved by improving the oxygen conditions, without drastically affecting the physiological condition of the yeast and beer quality.     

Isolation and Characterization of Brewer's Yeast Variants with Improved Fermentation Performance under High-Gravity Conditions

To save energy, space, and time, today's breweries make use of high-gravity brewing in which concentrated medium (wort) is fermented, resulting in a product with higher ethanol content. After fermentation, the product is diluted to obtain beer with the desired alcohol content. While economically desirable, the use of wort with an even higher sugar concentration is limited by the inability of brewer's yeast (Saccharomyces pastorianus) to efficiently ferment such concentrated medium. Here, we describe a successful strategy to obtain yeast variants with significantly improved fermentation capacity under high-gravity conditions. We isolated better-performing variants of the industrial lager strain CMBS33 by subjecting a pool of UV-induced variants to consecutive rounds of fermentation in very-high-gravity wort (>22° Plato). Two variants (GT336 and GT344) showing faster fermentation rates and/or more-complete attenuation as well as improved viability under high ethanol conditions were identified. The variants displayed the same advantages in a pilot-scale stirred fermenter under high-gravity conditions at 11°C. Microarray analysis identified several genes whose altered expression may be responsible for the superior performance of the variants. The role of some of these candidate genes was confirmed by genetic transformation. Our study shows that proper selection conditions allow the isolation of variants of commercial brewer's yeast with superior fermentation characteristics. Moreover, it is the first study to identify genes that affect fermentation performance under high-gravity conditions. The results are of interest to the beer and bioethanol industries, where the use of more-concentrated medium is economically advantageous.     

Physiological characterization of brewer’s yeast in high-gravity beer fermentations with glucose or maltose syrups as adjuncts

High-gravity brewing, which can decrease production costs by increasing brewery yields, has become an attractive alternative to traditional brewing methods. However, as higher sugar concentration is required, the yeast is exposed to various stresses during fermentation. We evaluated the influence of high-gravity brewing on the fermentation performance of the brewer’s yeast under model brewing conditions. The lager brewer’s strain Weihenstephan 34/70 strain was characterized at three different gravities by adding either glucose or maltose syrups to the basic wort. We observed that increased gravity resulted in a lower specific growth rate, a longer lag phase before initiation of ethanol production, incomplete sugar utilization, and an increase in the concentrations of ethyl acetate and isoamyl acetate in the final beer. Increasing the gravity by adding maltose syrup as opposed to glucose syrup resulted in more balanced fermentation performance in terms of higher cell numbers, respectively, higher wort fermentability and a more favorable flavor profile of the final beer. Our study underlines the effects of the various stress factors on brewer’s yeast metabolism and the influence of the type of sugar syrups on the fermentation performance and the flavor profile of the final beer.     

Isolation and characterization of brewer's yeast variants with improved fermentation performance under high-gravity conditions

To save energy, space, and time, today's breweries make use of high-gravity brewing in which concentrated medium (wort) is fermented, resulting in a product with higher ethanol content. After fermentation, the product is diluted to obtain beer with the desired alcohol content. While economically desirable, the use of wort with an even higher sugar concentration is limited by the inability of brewer's yeast (Saccharomyces pastorianus) to efficiently ferment such concentrated medium. Here, we describe a successful strategy to obtain yeast variants with significantly improved fermentation capacity under high-gravity conditions. We isolated better-performing variants of the industrial lager strain CMBS33 by subjecting a pool of UV-induced variants to consecutive rounds of fermentation in very-high-gravity wort (>22 degrees Plato). Two variants (GT336 and GT344) showing faster fermentation rates and/or more-complete attenuation as well as improved viability under high ethanol conditions were identified. The variants displayed the same advantages in a pilot-scale stirred fermenter under high-gravity conditions at 11 degrees C. Microarray analysis identified several genes whose altered expression may be responsible for the superior performance of the variants. The role of some of these candidate genes was confirmed by genetic transformation. Our study shows that proper selection conditions allow the isolation of variants of commercial brewer's yeast with superior fermentation characteristics. Moreover, it is the first study to identify genes that affect fermentation performance under high-gravity conditions. The results are of interest to the beer and bioethanol industries, where the use of more-concentrated medium is economically advantageous.     

Development of engineered yeast for biosorption of beer haze-active polyphenols

Compared to most other alcoholic beverages, the shelf life of beer is much more limited due to its instability in the bottle. That instability is most likely to appear as turbidity (haze), even sedimentation, during storage. The haze in beer is mostly caused by colloidal particles formed by interactions between proteins and polyphenols within the beer. Therefore, beers are usually stabilized by removing at least one of these components. We developed and constructed a Saccharomyces cerevisiae strain with a proline-rich QPF peptide attached to the cell wall, using the C-terminal anchoring domain of α-agglutinin. The QPF peptide served to bind polyphenols during fermentation and, thus, to decrease their concentration. Strains displaying QPF were able to bind about twice as much catechin and epicatechin as a control strain displaying only the anchoring domain. All these experiments were done with model solutions. Depending on the concentration of yeast, uptake of polyphenols was 1.7–2.5 times higher. Similarly, the uptake of proanthocyanidins was increased by about 20 %. Since the modification of yeasts with QPF did not affect their fermentation performance under laboratory conditions, the display of QPF appears to be an approach to increase the stability of beer.

     

Continuous beer fermentation by high cell-density culture of bottom brewer's yeast

Continuous beer production was investigated in a high cell-density culture system which consisted of two stages for the fermentation and sedimentation of yeast cells. The continuous culture was carried out for a fermentation time of 5,500 h without contamination, at varying dilution rates and fermentation temperatures in the ranges of 0.017-0.033 h⁻¹ and 6.5–8.5°C, respectively. This process was found to be suitable for continuous and stable beer brewing. Under these conditions, the cell concentration in the first stage was about 80 times as high as that in the exit of the second stage. Concentrations of viable cells, sugar and ethanol were maintained at 1.3 × 10⁹ cells/ml, 25 and 36 g/l, respectively, and were hardly affected by fermentation temperature. Concentrations of ethyl acetate, isoamyl alcohol and isoamyl acetate were similar in the fermentation temperature ranges of 6.5–8.5°C, and the amounts at a fermentation temperature of 7°C were comparable to those of lager-type beer. Diacetyl flavor, which is known to be an effluent component that causes deterioration in the second stag e (young beer), was maintained at 1.2 ppm at a dilution rate and fermentation temperature of 0.022 h⁻¹ and 7°C, respectively. The diacetyl flavor was due to the accumulation of vicinal diketone, the precursor of which is acetohydroxy acid. The acetohydroxy acid was converted to vicinal diketone by pretreatment at 60°C for 30 min. The vicinal diketone was then consumed by the yeast during after-fermentation at a fermentation temperature of 3°C. Using this method, total vicinal diketone decreased below 0.3 ppm for an after-fermentation time of 6.8 h, which was 225 times as fast as that of after-fermentation without the pretreatment. This process may make it possible to achieve continuous beer fermentation from the fermentation stage to after-fermentation for diacetyl removal.     

High-Gravity Brewing: Effects of Nutrition on Yeast Composition, Fermentative Ability, and Alcohol Production

A number of economic and product quality advantages exist in brewing when high-gravity worts of 16 to 18% dissolved solids are fermented. Above this level, production problems such as slow or stuck fermentations and poor yeast viability occur. Ethanol toxicity has been cited as the main cause, as brewers' yeasts are reported to tolerate only 7 to 9% (vol/vol) ethanol. The inhibitory effect of high osmotic pressure has also been implicated. In this report, it is demonstrated that the factor limiting the production of high levels of ethanol by brewing yeasts is actually a nutritional deficiency. When a nitrogen source, ergosterol, and oleic acid are added to worts up to 31% dissolved solids, it is possible to produce beers up to 16.2% (vol/vol) ethanol. Yeast viability remains high, and the yeasts can be repitched at least five times. Supplementation does not increase the fermentative tolerance of the yeasts to ethanol but increases the length and level of new yeast cell mass synthesis over that seen in unsupplemented wort (and therefore the period of more rapid wort attenuation). Glycogen, protein, and sterol levels in yeasts were examined, as was the importance of pitching rate, temperature, and degree of anaerobiosis. The ethanol tolerance of brewers' yeast is suggested to be no different than that of sake or distillers' yeast.     

Effect of hot trub and particle addition on fermentation performance of Saccharomyces cerevisiae

In this investigation, the effect of hot trub (a precipitation product of the wort boiling process in beer manufacturing) addition on fermentation performance was observed under variation of yeast vitality, and origin and the amount of hot trub. Its addition improved suspended cell concentrations for all yeast vitalities tested, and the more trub was added, the greater the effect. Further, pilot-scale fermentations showed significantly lower pH values and an accelerated extract degradation, thus, advancing fermentation by roughly 1 day for hot trub addition versus the fermentation of extremely bright wort. Since the positive effect of trub has often been associated with its particulate characteristics, fermentations with fractionated model particles, such as poly(vinylpyrrolidones) and kieselguhr, of different particle sizes were carried out under variation of yeast vitality and particle amounts. The addition of both particle types also improved fermentation performance, however, the effect was not as great as that of hot trub. Particulate material may improve the development of CO₂ from the fermenting medium, thus reducing its concentration and inhibitory effect on yeast metabolism. The most effective fraction of kieselguhr had a 40 μm peak which also occurred in particle size distributions of all hot trubs investigated. This could be of particular interest when discussing particle effects.     

Filtration, haze and foam characteristics of fermented wort mediated by yeast strain

AIMS: To investigate the influence of the choice of yeast strain on the haze, shelf life, filterability and foam quality characteristics of fermented products. METHODS AND RESULTS: Twelve strains were used to ferment a chemically defined wort and hopped ale or stout wort. Fermented products were assessed for foam using the Rudin apparatus, and filterability and haze characteristics using the European Brewing Convention methods, to reveal differences in these parameters as a consequence of the choice of yeast strain and growth medium. CONCLUSIONS: Under the conditions used, the choice of strain of Saccharomyces cerevisiae effecting the primary fermentation has an impact on all of the parameters investigated, most notably when the fermentation medium is devoid of macromolecular material. SIGNIFICANCE AND IMPACT OF THE STUDY: The filtration of fermented products has a large cost implication for many brewers and wine makers, and the haze of the resulting filtrate is a key quality criterion. Also of importance to the quality of beer and some wines is the foaming and head retention of these beverages. The foam characteristics, filterability and potential for haze formation in a fermented product have long been known to be dependant on the raw materials used, as well as other production parameters. The choice of Saccharomyces cerevisiae strain used to ferment has itself been shown here to influence these parameters.     

Improved thin-layer chromatography separation of carbohydrates in wort and beer

Summary With a slight modification the method previously reported by Vrbaški and Lepojević,J. Chromatog. 558, 328–332 (1991) can be applied to the analysis of carbohydrates in worts, beer and brewing syrup. The modification introduces new a third step to the development of plates using chloroform/glac. acetic acid/water (2;6;2 by vol). By this method it is possible to separate maltooligosaccharides up to 17 spots. Evidence of a difference between the yeast strains in fermentation of carbohydrate is also presented.     

Influence of valine and other amino acids on total diacetyl and 2,3-pentanedione levels during fermentation of brewer’s wort

Undesirable butter-tasting vicinal diketones are produced as by-products of valine and isoleucine biosynthesis during wort fermentation. One promising method of decreasing diacetyl production is through control of wort valine content since valine is involved in feedback inhibition of enzymes controlling the formation of diacetyl precursors. Here, the influence of valine supplementation, wort amino acid profile and free amino nitrogen content on diacetyl formation during wort fermentation with the lager yeast Saccharomyces pastorianus was investigated. Valine supplementation (100 to 300 mg L^?1) resulted in decreased maximum diacetyl concentrations (up to 37 % lower) and diacetyl concentrations at the end of fermentation (up to 33 % lower) in all trials. Composition of the amino acid spectrum of the wort also had an impact on diacetyl and 2,3-pentanedione production during fermentation. No direct correlation between the wort amino acid concentrations and diacetyl production was found, but rather a negative correlation between the uptake rate of valine (and also other branched-chain amino acids) and diacetyl production. Fermentation performance and yeast growth were unaffected by supplementations. Amino acid addition had a minor effect on higher alcohol and ester composition, suggesting that high levels of supplementation could affect the flavour profile of the beer. Modifying amino acid profile of wort, especially with respect to valine and the other branched-chain amino acids, may be an effective way of decreasing the amount of diacetyl formed during fermentation.     

Applicability of Yeast Extracellular Proteinases in Brewing: Physiological and Biochemical Aspects

A general screening survey for expression of extracellular acid proteinase production was performed on over 100 cultures belonging to the genus Saccharomyces. Although two strains of Saccharomyces cerevisiae showed positive extracellular proteinase phenotypes in plate tests, it was not possible to demonstrate proteolytic activities in cell-free culture supernatants in assays performed at beer pH values. Of several yeasts from other genera examined, Saccharomycopsis fibuligera and Torulopsis magnoliae produced extracellular proteinases with desirable properties. Proteolytic activities were detected in assays performed at beer pH values and at lower temperature. Brewer's wort served as a highly inducing medium for extracellular proteinase production, with T. magnoliae yielding enzyme of highest specific activity. In fact, commencement of enzyme production was detected shortly after the onset of exponential growth in brewer's wort. Inclusion of crude enzyme preparations in brewer's wort inoculated simultaneously with brewer's yeast reduced final ethanol yields slightly and was found to be effective in reducing chill haze formation in bottled beer.     

Surface-<Emphasis Type="Italic">engineered Saccharomyces cerevisiae</Emphasis> displaying α-acetolactate decarboxylase from <Emphasis Type="Italic">Acetobacter aceti</Emphasis> ssp <Emphasis Type="Italic">xylinum</Emphasis>

Objectives

To convert α-acetolactate into acetoin by an α-acetolactate decarboxylase (ALDC) to prevent its conversion into diacetyl that gives beer an unfavourable buttery flavour.

Results

We constructed a whole Saccharomyces cerevisiae cell catalyst with a truncated active ALDC from Acetobacter aceti ssp xylinum attached to the cell wall using the C-terminal anchoring domain of α-agglutinin. ALDC variants in which 43 and 69 N-terminal residues were absent performed equally well and had significantly decreased amounts of diacetyl during fermentation. With these cells, the highest concentrations of diacetyl observed during fermentation were 30 % less than those in wort fermented with control yeasts displaying only the anchoring domain and, unlike the control, virtually no diacetyl was present in wort after 7 days of fermentation.

Conclusions

Since modification of yeasts with ALDC variants did not affect their fermentation performance, the display of α-acetolactate decarboxylase activity is an effective approach to decrease the formation of diacetyl during beer fermentation.